The determination of nucleic acids has become an important tool in analytical chemistry, especially in health care. For example, infection diseases and genetic status can be easily determined on the basis of the presence or the amount of a nucleic acid indicative of said disease or status in samples received from the individual. For this reason methods were established using sequence specific hybridization of a nucleic acid, preferably an oligonucleotide, with a target nucleic acid indicative for that disease or genetic status. Sensitive techniques like the branched DNA-method (U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697), or methods detecting rRNA target nucleic acids (EP 0 272 009), which are present in high copy numbers in an organism, can be used for direct detection of a target nucleic acid in a sample from that organism. But many target nucleic acids are present in an organism in such low concentration, that a direct detection in a sample derived from that organism is not possible. Such targets need to be amplified before detection. Suitable amplification methods are for example LCR (U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; WO 90/01069; WO 89/12696; and WO 89/09835), cycling probe technology (U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO 95/00667), Invader TM technology (U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669), Q-Beta replicase technology (U.S. Pat. No. 4,786,600), NASBA (U.S. Pat. No. 5,409,818; EP-0 329 822), TMA (U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365, 5,710,029), SDA (U.S. Pat. Nos. 5, 455,166 and 5,130,238) and PCR (U.S. Pat. No. 4,683,202).
In order to minimize false results in nucleic acid determinations, authorities in several countries require the use of control nucleic acids. Especially when using amplification methods such control nucleic acids are very important, because the amplification process can be strongly influenced by the reaction conditions, which could lead to misleading results. Sometimes inhibitory substances are contained in a sample, which could lead to false negative results.
In general one can distinguish external and internal controls. External controls, like classical positive and negative controls are normally used to check whether the assay runs properly or whether contaminants are contained. An internal control for example is useful for recognizing inhibitory substances possibly contained in a sample or can be used as a quantification standard in a quantitative assay. In contrast to an external control, which normally is tested in a separate reaction chamber, an internal control is preferably incubated in the same reaction chamber together with the analyte to be tested. Therefore, the control or the amplified product of that control has to be distinguishable from the analyte or from the amplified product of that analyte. When using an amplification method an internal control nucleic acid is being co-amplified essentially under the same reaction conditions as the target nucleic acid. These conditions include reagent concentrations, temperature, inhibitor concentration or enzymatic activities. Frequently used sequences for controls are derived from housekeeping genes (see Chelly et al. (1990) Eur. J. Biochem. 187:691-698; Mallet et al. (1995) J. Clin. Microbiol. 33:3201-3208), but also non-natural sequences are being used (Besnard et al. (1995) J. Clin. Microbiol. 32:1887-1893; Gilliland et al. (1990) Proc. Natl. Acad. Sci. USA 87:2725-2729; Wang et al. (1989) Proc. Natl. Acad. Sci. USA 9717-9721).
The amplified nucleic acid derived from the internal control can be distinguished from the amplified nucleic acid derived from the target nucleic acid for example by their different length or hybridization capability to a distinct probe (for reviews see: Clementi et al. (1990) PCR Methods Applic. 2:191-196; Clementi et al. (1995) Arch. Virol. 140:1523-1539). In all cases the nucleotide sequence of the internal control is partially or totally different from the target nucleic acid sequence. However the sequence and the length of a nucleic acid determine its GC-content, secondary structures and melting temperature and, therefore, is essential for its behavior in a hybridization and amplification reaction. A different sequence of an internal control in nearly all cases result in a different behavior of the control nucleic acid compared with that of the target nucleic acid. In contrast thereto an ideal internal control should mimic exactly the target nucleic acid in order to allow a proper monitoring of the reaction.
One of the most critical aspects in an amplification reaction is the binding of the primer to the target nucleic acid. Therefore internal controls are being used, which have the same primer binding sites as the target nucleic acid (see for example Gilliland et al. (1990) Proc. Natl. Acad. Sci. USA 87:2725-2729; Wang et al. (1989) Proc. Natl. Acad. Sci. USA 9717-9721; U.S. Pat. No. 5,219,727). This could lead to a competition in the reaction for the primers and could result in a decreased sensitivity of the assay.
Gilliland et al. (1990) Proc. Natl. Acad. Sci. USA 87:2725-2729 describe internal controls which nearly have the same nucleotide sequence as the target nucleic acid, but contain a new restriction enzyme cleavage site or the sequence of an intron region not contained in the target nucleic acid. Due to the very high homology of the internal control with the target, both nucleic acids as well as the amplificates can cross-hybridize with each other. Dependent on the detection method used, this can lead to wrong results especially in quantitative assays. Also, the described methods requires elaborous techniques like restriction enzyme digestion and agarose gel electrophoresis of the amplified products.
US patent application No. US 2001/0006800 A1 describes related control nucleic acids which comprise the internal target sequence without the primer regions in an inverted orientation. This application does not describe controls which comprise the complementary target sequence in an inverted orientation nor controls comprising target primer sequences in an inverted orientation. Especially it is to note that inversion of the primer sequences would lead to sequences which do have different Tm's and GC-contents compared to the original primer sequences.
It is an object of the present invention to improve the methods for determination of nucleic acids, especially in avoiding all or a part of the disadvantages of the known methods.